The separation of biopolymers such as proteins, polynucleotides, carbohydrates and peptides is important in the purification and analysis of such materials, in facilitating biochemical investigations, in medical testing and diagnosis, and in the genetic engineering/biotechnology fields.
In the past, such materials have been separated by traditional chromatographic techniques, including size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) on gel-type chromatographic supports. Such systems provide some resolution, but separation times are long because only low pressure can be used for mobile phase flow. In particular, the supporting gels cannot be subjected to high pressures to speed the flow of mobile phase.
Some attempts have been made to use modern high performance reversed phase liquid chromatography (RPLC) for the separation of such biopolymeric materials, to take advantage of the high speed and high resolution afforded by the sturdy small-particle silica-based chromatographic column packing materials employed in this technique. These attempts have shown some limited success, but have generally failed with mixtures of biopolymers because the biological materials either are irreversibly adsorbed to the strongly-hydrophobic stationary phases presently in use, e.g. n-alkyl, or can be removed only by mobile phases containing polar organic solvents and/or organic acids. Such harsh conditions frequently disrupt the quaternary, tertiary, and/or secondary structures of biopolymers, causing denaturation which destroys biological activity. They can also degrade chromatographic column packings.
Various attempts have been made to synthesize new chromatographic packing materials having stationary phases which bind biopolymers only weakly, so that they can be eluted under mild conditions which do not cause denaturation of the polymers or degradation of the chromatographic column packings. Covalently bonded stationary phases have been constructed using glycidyl ethers, glycidoxysilane derivatives, mixtures of glycidoxysilane derivatives and alkylsilanes, short-chain alkylsilanes, variously acylated polyaziridine, and ether-substituted silanes. All of these prior art phases suffer from one or more of the following deficiencies: they contain reactive and/or charged functional groups in the chromatographically-involved portion of the molecules, groups which can interact chemically with eluting solvents or with solutes being separated; they contain easily hydrolyzable linkages, and are therefore not chemically stable; they are too hydrophobic,and require polar organics for elution of proteins, conditions which are known to cause denaturation; and their hydrophobicity is difficult to adjust and control reproducibly.
An example of a silyl ether stationary phase precursor is Cl.sub.2 (CH.sub.3)Si--(CH.sub.2).sub.3 (CH.sub.2 CH.sub.2 O).sub.3 CH.sub.3, disclosed by O. Schou and P. Larsen in Acta. Chem. Scand., B 35, 337 (1981). This compound possesses on the silicon atom both a methyl group and a chain of five methylene (CH.sub.2) units before the first oxygen atom, and thus when bonded to silica it produces a stationary phase too hydrophobic for hydrophobic interaction chromatographic separations of biopolymers such as proteins. This is shown in the reference by the author's use of the polar organic solvents methanol and tetrahydrofuran to elute a peptide mixture from a chromatographic column prepared with this bonded packing material.
Several approaches have been taken in designing and producing weakly hydrophobic silica-based bonded stationary phase chromatographic packings. In the simplest case, bonded stationary phases consisting of a single type of ligand are produced by one of two general bonding procedures to be discussed below. Although this is simple and straightforward, the hydrophobicity of the resulting bonded phases is limited by the availability of only a finite number of stationary phase precursors. Bonded phases containing mixtures of ligands have also been employed to balance and blend the properties of the several ligands to produce surface phases optimized for particular chromatographic purposes. Such mixed phases have been produced in two ways: by bonding a selected mixture of ligand precursors to silica in a single bonding step, or by a series of bonding steps and derivatization reactions which introduce various ligands and functionalities sequentially. The more popular sequential approach to bonded phase construction permits a variety of bonded phases to be produced, but is inherently irreproducible since organic reactions seldom go to completion or to precisely reproducible extents of reaction. Thus, if two or more successive reactions are employed to create a mixed bonded phase, later repetition of the sequence will produce a similar but non-identical mixed bonded phase.
The ligand precursors most commonly used in the prior art preparation of chemically bonded stationary phases have been glycidyl ethers, ##STR1## and reactive silanes which contain one to three halogen or alkoxyl groups on the silicon atom. The glycidyl ethers and monofunctional silanes can form only a single covalent bond with silica, thus producing bonded layers having inherently low stability. The bifunctional silanes create bonded layers of somewhat higher stability since they have the capacity to form more chemical bonds. Trifunctional silanes can, in principle, form the greatest number of bonds to the silica surface and hence would be expected to produce the most stable bonded coatings. However, trifunctional silanes have too frequently reacted irreproducibly to give bonded phases having low chromatographic utility as a result of having an excessively high loading of bonded phase as well as poor stabilities under chromatographic conditions. These problems are a result of uncontrolled polymerization and cross-linking of trifunctional silanes in the presence of excess water, a topic which will be discussed below.
Two general bonding procedures have usually been employed to bond organosilanes to silica, depending on the sort of bonded layer to be produced. If the reaction between silica and organosilane is carried out under strictly anhydrous conditions, where the silica is dried by applying heat and vacuum and then refluxed with the organosilane in sodium-dried solvent, a monolayer of chemically bonded stationary phase is obtained. On the other hand, if the reaction between silica and a bifunctional or trifunctional organosilane is conducted in the presence of even a trace of water, some polymerization of the silane occurs, presumably via hydrolysis of some of the reactive functionalities of the silane to yield silanol groups which in turn react with additional organosilane reagent. Thus, if high loadings of bonded stationary phase are desired, the bonding procedure is conducted in the presence of water. FIG. 1 illustrates some of the sorts of structures which can form when a dialkoxy silane reacts with silica in the presence of water; all structures shown are present. The situation in the case of a trifunctional silane is similar but more complex. Such polymerization of organosilanes has in general been considered a problem, however, because it is frequently irreproducible and can cause overloading of the silica with bonded phase, thereby producing chromatographic packing materials having low surface area and poor porosity. In addition, bonded layers can be produced which are extensively polymerized but not extensively bonded to silica, with the result that if some of the silane-to-silica bonds are hydrolyzed, large patches of bonded phase can be lost and correspondingly large patches of unprotected silica surface can be exposed. Such stationary phases have been found to degrade rapidly and irreproducibly and to give irreproducible chromatography. As a result of these problems with uncontrolled polymerization and cross-linking when using trifunctional organosilanes, many workers have preferred, where possible, to use the mono or difunctional organosilanes in which such polymerization is either impossible or limited.
Two examples of atypical methods for bonding reactive organosilanes to silica are given by Larsson, and Majors. P. Larsson et al., Advances in Chromatography, 21, 41 (1983); R. Majors and M. Hopper, J. Chromat. Sci., 12, 767, (1974). Larsson et al. refluxed a trialkoxy silane with carefully dried silica and a trace of triethylamine in carefully dried toluene. This procedure gives a monolayer of silane bonded to silica, with minimal polymerization and cross-linking, but creates the possibility of hydrolysis of unreacted alkoxy groups in subsequent handling or use of the packing material in the presence of water, with concomitant uncontrolled polymerization of silane at the silica surface and irreproducible chromatography. Majors equilibrated silica with water vapor in an atmosphere of low constant relative humidity, then slurried this with trialkoxysilane in dry toluene and slowly raised the temperature to the boiling point to complete the reaction. His procedure suffers in producing bonding reactions having relative standard deviations in reproducibility of surface converage as high as nine percent, and his bonded stationary phases are of undisclosed stability under chromatographic conditions. Further, both of the above processes employ a solvent, and differ from the process of the present invention in several respects.
In view of the above, it is clear that if biopolymer separations are to be performed in modern high performance liquid chromatographic instruments, by hydrophobic interaction chromatography and size exclusion, improved chromatographic packing materials are needed. Such improved packings in turn require new stationary phase precursors capable of providing stable bonded layers of stationary phases which are non-reactive and only weakly hydrophobic, and further require the development of improved bonding procedures which reproducibly yield uniform and stable layers of bound stationary phases.